Plant biomass is the most abundant, natural and renewable resource on earth. Cellulose is the major constituent of plant cell walls and is a commercially important raw material that can be converted into useful products with appropriate methodologies (Hon DN Cellulose: a random walk along its historical path, In Cellulose. J. C. Roberts (ed.), Chapman & Hall, 1-25, 1994). One such application is the production of fermentable sugars from cellulose for the production of bio-fuel ethanol. Because of the potentially high importance of this process, cellulose-degrading enzymes have received a great deal of attention (Beltrane P L et al., Bioresource Technol. 39: 165-171, 1992). To date, the conversion of plant cellulose into fermentable sugars is accomplished with commercial enzymes produced by large-scale fermentation. However, the cost of producing these enzymes remains a significant barrier to the widespread utilization of this process (Cowan D, Tibtech. 14: 177-178, 1996; Ho N W et al., Adv. Biochem. Eng. Biotechnol. 65: 163-192, 1999). Thus, various approaches have been taken to produce cellulose-degrading enzymes on a large scale and at low cost (Goddijin O J M and Pen J, Trends biotechnol. 13: 3790-3870, 1995; Gidding G et al., Nature Biotech. 18: 1151-1155, 2000). Trichoderma reesei has been a target strain for the production of cellulase, and Bacillus and Aspergillus microorganisms have been a target for the production of xylanase. But, the concentration and activity of such enzyme is not enough to be used in industry. High production costs are also a problem, resulted from a high priced purified medium containing expensive materials such as lactose as a substrate for the production of the enzyme.
Genetically engineered transgenic plants are one of the most economical systems for large scale production of recombinant proteins for industrial and medical uses, because a large quantity of enzymes can be produced with minimal input. In efforts to lower the production costs of cellulases (Jensen L G et al., Proc. Natl. Acad. Sci. 93: 3487-3491, 1996; Kawazu T et al., J. Ferment. Bioeng. 82: 205-209, 1996; Nuutila A M et al., Plant Mol. Biol. 41: 777-783, 1999; Ziegelhoffer T et al., Mol. Breed. 5: 309-318, 1999; Dai Z et al., Transgenic Res. 9: 43-54, 2000), the cellulase gene has been expressed in transgenic plants, but the expression levels obtained have been low. The purification of recombinant cellulases expressed in plants is also an expensive step, which contributes greatly to the cost of production of fermentable sugars from cellulose.
Thus, the present inventors produced transgenic plants in which a recombinant gene containing cellulase gene, cellulose binding domain gene and chloroplast targeting gene was inserted. And the present inventors completed this invention by confirming that the conversion of cellulose into fermentable sugar is possible in transgenic plants themselves by autohydrolysis using cellulose localized in chloroplasts, and the production cost is also reduced by simplifying the production line of soluble sugar from cellulose.